Briefly, total RNA was extracted from RPTCs using TRIzol® reagent (Invitrogen Life Technologies), according to the manufacturer's instructions. Next, 5 μg of total RNA was used to prepare cDNA. A reverse transcriptase kit (Superscript® III, Invitrogen Life Technologies) was used for cDNA synthesis, at 55°C for 50 min, followed by 85°C for 5 s, on an ABI GeneAmp® PCR System 9700 (Applied Biosystems). The transcripts were quantified using the ABI 9700 and SYBR® Premix reagent (Thermo Fisher Scientific), according to the manufacturer's instructions. Reactions began with a 10-s hot-start activation of the Taq polymerase at 95°C, followed by 45 cycles of amplification in 3 steps (denaturation at 95°C for 5 s, followed by 30 s annealing at 60°C, and 30 s extension at 72°C). The primers used in each reaction are shown in Table 1 . Data are expressed as the mean ± standard deviation of 4 independent experiments.
Abi 9700
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 9700 is a thermal cycler instrument designed for nucleic acid amplification and analysis. It is capable of performing polymerase chain reaction (PCR) protocols. The core function of the ABI 9700 is to precisely control the temperature and cycling conditions required for various PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Taq polymerase, by Thermo Fisher Scientific (2 mentions)
Dntps, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Abi geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Agencourt ampure xp kit, by Beckman Coulter (1 mentions)
Rneasy mini kit, by Thermo Fisher Scientific (1 mentions)
Antibody to β actin, by Boster Bio (1 mentions)
Abi 7500, by Thermo Fisher Scientific (1 mentions)
β actin, by Boster Bio (1 mentions)
Bigdye terminator mix v3, by Thermo Fisher Scientific (1 mentions)
Exosap it pcr product cleanup kit, by Thermo Fisher Scientific (1 mentions)
Abi 3730xl, by Thermo Fisher Scientific (1 mentions)
Premix taq, by Takara Bio (1 mentions)
Epitect kit, by Qiagen (1 mentions)
Hotstart taq, by Takara Bio (1 mentions)
Pyromark q96 id system, by Qiagen (1 mentions)
28 protocols using abi 9700
1
Quantitative RT-PCR for Gene Expression
2
HPV DNA Genotyping with Luminex
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Total DNA was extracted from 200 µl exfoliated cervical cell suspension with the HPV DNA extraction kit (TELLGEN, Shanghai, China) according to the manufacturer's instructions. Using the HPV DNA genotyping kit (TELLGEN, Shanghai, China), the Luminex® xMAP™ technology can quickly and quantitatively detect 27 types of HPV (high risk subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56 , 58, 59, 66, 68, 26, 53, 82, and low risk subtypes 6, 11, 40, 42, 43, 44, 55, 61, 81, 83). Briefly, 5 µl extracted DNA solution was used for PCR in a reaction (20 µl) containing 10 µl PCR mixture, 5 µl primer mixture, and 0.8 µl rTaq polymerase. The PCR amplification was performed and 3 µl products were added to microsphere hybrid for hybridization procedure on an ABI9700 (ThermoFisher, Waltham, U.S.A.). Each reaction pore was added 75 µl SA-PE. The mixture was detected on Luminex200 multifunctional flow detector (R&D system, Minneapolis, U.S.A.).
3
Fecal Bacterial DNA Extraction and 16S rRNA Sequencing
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Bacterial DNA was extracted from 1 g of fecal sample using the OMEGA kit (Omega Bio-Tek, Norcross, GA, USA), following the manufacturer’s instructions. DNA concentration and purity were evaluated using the Nanodrop 2000 Spectrophotometer (Thermo Scientific, Waltham, USA). To amplify the V3–V4 region of the bacterial 16S rRNA gene, primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′, forward) and 806R (3′-GGACTACNNGGGTATCTAAT-5′, reverse) were used. The following PCR amplification program was used: 5-min denaturation at 95°C; 28 cycles at 95°C for 45 s, 55°C for 50 s, and 72°C for 45 s; and a final extension at 72°C for 10 min. The amplified fragments were identified using 2% (w/v) agarose gel electrophoresis, purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics, Indianapolis, IN, USA), and quantified using PCR (ABI 9700; Thermo Fisher Scientific, Waltham, MA, USA). The purified PCR products were sequenced following standard protocols, using a 2 × 250 bp sequencing kit and Illumina MiSeq (Illumina, San Diego, CA, USA).
4
Genetic diversity analysis of peanut genotypes
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Progenies and parents were analyzed by microsatellite markers (Table 4 ) developed for A. hypogaea (Moretzsohn et al., 2013 ). Total genomic DNA was extracted from young and fresh leaflets of 41 genotypes, including parents and progenies individuals, according to the method of Doyle and Doyle (1990). The amount and quality of the DNA were evaluated by 1% agarose gel electrophoresis. PCR assays were run with 0.2 μl DNA Taq polymerase (5 U/μL), 0.5 μL buffer (with Mg), 1.0 μL dNTPS (2.5 nM), 1.0 μL ultrapure water, 1.2 μL BSA (2.5 mg/mL), 0.1 μL of each primer (10 μm) and 2.0 μL genomic DNA, in a final volume of 6 μL. Amplification reactions were performed in an ABI 9700 (Applied Biosystems, Foster City, CA, USA) thermal cycler, under the following conditions: 94 °C for 5 min, followed by 30 cycles at 94 °C for 1 min, 58 °C for 1 min (depending on annealing temperature of the primer), 72 °C for 1 min, and final extension at 72 °C for 1 min. The allelic detection of 30 SSR loci was performed in an ABI377 automated sequencer in a multiplex loci system (Table 4 ). Genetic diversity was analyzed by the PowerMarker V 3.25 and NTSYS programs.
5
Retinal RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from retina using Tri-reagent. Isolated RNA was further purified by RNeasy Mini Kit and quantified by measuring the absorbance at 260 and 280 nm on a spectrophotometer (NanoDrop Technologies, Delaware). Two micrograms of total RNA were reverse transcribed using a High Capacity cDNA Reverse Transcription Kit. The reverse transcription reaction was performed using a thermocycler (ABI 9700, Applied Biosystems, Foster City, CA), and reaction conditions were as follows: initial reverse transcription for 10 min at 25 °C, followed by 37 °C for 120 min and 84 °C for 5 min. Real-time PCR (RT-PCR; Applied Biosystems) was performed in triplicate with 25 ng of cDNA template using SYBR Green Master Mix with gene-specific primers (Appendix 1). The reaction conditions were as follows: 40 cycles of initial denaturation temperature at 95 °C for 30 s, followed by annealing at 52 °C for 40 s, and extension at 72 °C for 1 min. The product specificity was analyzed using melt curve analysis. Normalization and validation of data were performed using β-actin as an internal control, and data were compared between WNIN/GR-Ob and the age-matched lean samples according to the comparative threshold cycle (2-ΔΔct ) method. Student t tests were used to compare the mean values among different groups for a given age. Values of p<0.05 were considered significant.
6
Quantifying Apoptosis Markers in HeLa Cells
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The relative levels of p53, Bax, Bcl-2 or caspase-3 mRNA in HeLa were examined by RT-PCR (SYBR Green) (ABI 3900, High-Throughput DNA/RNA Synthesizer; ABI 9700, PCR instrument; ABI 7500, fluorescent quantitative PCR instrument; Applied Biosystems, USA), as shown in Fig. 4D . The HeLa cells were treated by four OA-MNPs groups (the OA-MNPs-a, OA-MNPs-b, OA-MNPs-c, and OA-MNPs-d) for 48 hours. The following primers of p53, Bax, Bcl-2 or caspase-3 designed with Primer Express 2.0 Software were used for the PCR step: (1) p53, Bax, Bcl-2 or caspase-3 sense, 5′-CCGAGTGGAAGGAAATTTGC-3′, 5′-CATGTTT TCTGACGGCAACTTC-3′, 5′-TGGGATGCCTTTGTGGAACT-3′ or 5′-TACCAGTGGA GGCCGACTTC-3′; (2) p53, Bax, Bcl-2 or caspase-3 antisense, 5′-AGTCAGAGCCAA CCTCAGGC-3′, 5′-AGGGCCTTGAGCACCAGTTT-3′, 5′-GAGACAGCCAGGAGAAAT CAAAC-3′ or 5′-CAAAGCGACTGGATGAACCA-3′; (3) β-actin sense, 5′-GCGCGG CTACAGCTTCA-3′; and (4) β-actin antisense, 5′-TCTCCTTAATGTCACGCACGAT-3′. The gene levels were normalized by re-probing the blots with antibody to β-actin (Boster Biological Technology Co., Ltd., China), as shown in Fig. 4e .
7
Bacterial 16S rDNA Identification Protocol
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For molecular identification, bacterial DNA was extracted from overnight cultures (TSB medium, 37 °C) using the kit for the isolation of genomic DNA from bacteria EXTRACTME (DNA Gdańsk, BLIRT S.A, Poland). DNA concentrations were determined using a UV–Vis spectrophotometer (NanoDrop 2000). Fragments of 16S ribosomal DNA (rDNA) were amplified using the primers 27F (5-AGAGTTTGATCMTGGCTCAG-3) and 1492R (5-GGTTACCTTGTTACGACTT-3). An amplification reaction was performed in a thermocycler ABI 9700 (Applied Biosystems™, USA) with the use of thermostable polymerase OptiTaq (EURx, Poland). Subsequently, PCR products were purified using the ExoSAP-IT PCR Product Cleanup Kit (Affymetrix, Inc., USA). Direct sequencing of PCR products was performed using 341F (5-CCTACGGGAGGCAGCAG-3), 518R (5-GTATTACCGCGGCTGCTGG-3), and 928F (5-TAAAACTYAAAKGAATTGACGGG-3) primers, BigDye Terminator Mix v3.1 and genetic analyzer ABI3730xl (Applied Biosystems™, USA). From obtained sequences, it was assembled the contig and then consensus sequences were compared with known 16S rDNA genes at the National Center for Biotechnology Information (NCBI) BLAST database (Altschul et al. 1997 (link)). Obtained sequence was submitted to GenBank, and it was received the accession number.
8
SIRT1 rs3758391 Genotyping Protocol
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SIRT1 rs3758391 was analyzed through PCR–RFLP, as previously described [19 (link)]. The primer for rs3758391 was forward 5′-ACGCAGGTAATTGATGCAGT-3′ and reverse 5′-CGTGAGCTATCTAGCCGTTT-3′. The total reaction system was 50 μl, including 25 μl of Premix Taq (Takara, Dalian, China), 2 μl (0.2 mM) of upstream and downstream primers, respectively, 1 μg of DNA template, and 16 μl of ddH2O. PCR conditions were programmed on an ABI 9700 thermal cycler (Applied Biosystems, Carlsbad, CA, USA) as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s, followed by 72 °C for 7 min. The PCR product was digested by addition of NcoI (New England Biolabs, Beverly, MA). The enzyme-digested products were analyzed by gel electrophoresis on 2.5% agarose gels. To confirmed our results, randomly selected amplified DNA samples were examined by direct sequencing method, and the results were 100% concordant.
9
MGMT Promoter Methylation Assessment
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MGMT promoter methylation status was detected by DNA pyrosequencing as previously described [34 (link), 35 (link)]. Bisulfite DNA modification was performed using the EpiTect Kit (Qiagen). The following primers were used to amplify the MGMT promoter region: 5′-GTTTYGGATATGTTGGGATA-3′ (forward) and 5′-biotin-ACCCAAACACTCACCAAATC-3′ (reverse). The PCR analysis was performed in duplicate in a 40-μl reaction volume containing 0.5 μl each primer (using a 10-μM working solution), 4 μl 10 × buffer, 3.2 μl of 2.5 μM dNTP, 2.5 U hotstart Taq (Takara Bio, Madison, WI, USA), and 2 μl of 10 μM bisulphite-treated DNA. The reaction conditions were: 95 °C for 3 min; 40 cycles of 95 °C for 15 s, 52 °C for 30 s, and 72 °C for 30 s; and 72 °C for 5 min (ABI 9700; Applied Biosystems, Foster City, CA, USA). DNA was purified from total PCR products using QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) and subjected to pyrosequencing (PyroMark Q96 ID System; Qiagen) using the primer 5′-GGATATGTTGGGATAGT-3′ in accordance with the manufacturer’s instructions. The obtained methylation values were averaged across the seven tested CpG loci within the MGMT promoter. Samples were considered as having a methylated MGMT promoter if the average methylation was ≥10 %.
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MGMT promoter methylation status was detected by DNA pyrosequencing as previously described [34 (link), 35 (link)]. Bisulfite DNA modification was performed using the EpiTect Kit (Qiagen). The following primers were used to amplify the MGMT promoter region: 5′-GTTTYGGATATGTTGGGATA-3′ (forward) and 5′-biotin-ACCCAAACACTCACCAAATC-3′ (reverse). The PCR analysis was performed in duplicate in a 40-μl reaction volume containing 0.5 μl each primer (using a 10-μM working solution), 4 μl 10 × buffer, 3.2 μl of 2.5 μM dNTP, 2.5 U hotstart Taq (Takara Bio, Madison, WI, USA), and 2 μl of 10 μM bisulphite-treated DNA. The reaction conditions were: 95 °C for 3 min; 40 cycles of 95 °C for 15 s, 52 °C for 30 s, and 72 °C for 30 s; and 72 °C for 5 min (ABI 9700; Applied Biosystems, Foster City, CA, USA). DNA was purified from total PCR products using QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) and subjected to pyrosequencing (PyroMark Q96 ID System; Qiagen) using the primer 5′-GGATATGTTGGGATAGT-3′ in accordance with the manufacturer’s instructions. The obtained methylation values were averaged across the seven tested CpG loci within the MGMT promoter. Samples were considered as having a methylated MGMT promoter if the average methylation was ≥10 %.
10
Rapid Bacterial Resistance Gene Detection
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Extracted plasmid DNA were amplified by standard PCR. Oligonucleotide primers of the TEM, SHV and CTX-M genes used were TEM: F-5′-TCCGCTCATGAGACAATAACC-3″, R-3′-ATAATACCGCACCACATAGCAG: SHV: F-5′-CGCCTGTGTATTATCTCCCT-3″, R-3″ CGAGTAGTCCACCACCAGATCCT-5′, CTX-M: F-5′-CGCTTTGCGATGTGCAG-3″, R-3′-ACCGCGATATCGTTGGT-5′ on a ABI 9700 Applied Biosystems thermal cycler at a final volume of 50 microliters for 35 cycles. Amplicons were resolved on a 1.5% agarose gel at 120V for 35 minutes and visualized on a UV transilluminator.
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